Investigating the role of uncoupling of troponin I phosphorylation from changes in myofibrillar Ca(2+)-sensitivity in the pathogenesis of cardiomyopathy.

Contraction in the mammalian heart is controlled by the intracellular Ca2+ concentration as it is in all striated muscle, but the heart has an additional signalling system that comes into play to increase heart rate and cardiac output during exercise or stress. β-adrenergic stimulation of heart muscle cells leads to release of cyclic-AMP and the activation of protein kinase A which phosphorylates key proteins in the sarcolemma, sarcoplasmic reticulum and contractile apparatus. Troponin I (TnI) and Myosin Binding Protein C (MyBP-C) are the prime targets in the myofilaments. TnI phosphorylation lowers myofibrillar Ca2+-sensitivity and increases the speed of Ca2+-dissociation and relaxation (lusitropic effect).Recent studies have shown that this relationship between Ca2+-sensitivity and TnI phosphorylation may be unstable. In familial cardiomyopathies, both dilated and hypertrophic (DCM and HCM), a mutation in one of the proteins of the thin filament often results in the loss of the relationship (uncoupling) and blunting of the lusitropic response. For familial dilated cardiomyopathy in thin filament proteins it has been proposed that this uncoupling is causative of the phenotype. Uncoupling has also been found in human heart tissue from patients with hypertrophic obstructive cardiomyopathy as a secondary effect. Recently, it has been found that Ca2+-sensitizing drugs can promote uncoupling, whilst one Ca2+-desensitising drug Epigallocatechin 3-Gallate (EGCG) can reverse uncoupling.We will discuss recent findings about the role of uncoupling in the development of cardiomyopathies and the molecular mechanism of the process

Uncoupling of myofilament Ca2+-sensitivity from troponin I phosphorylation by mutations can be reversed by Epigallocatechin-3-Gallate.

AIMS: Heart muscle contraction is regulated via the β-adrenergic response that leads to phosphorylation of Troponin I (TnI) at Ser22/23, which changes the Ca(2+)-sensitivity of the cardiac myofilament. Mutations in thin filament proteins that cause Dilated Cardiomyopathy (DCM) and some mutations that cause Hypertrophic Cardiomyopathy (HCM) abolish the relationship between TnI phosphorylation and Ca(2+)-sensitivity (uncoupling). Small molecule Ca(2+)-sensitisers and Ca(2+)-desensitisers that act upon troponin alter the Ca(2+)-sensitivity of the thin filament but their relationship with TnI phosphorylation has never been studied before. METHODS AND RESULTS: Quantitative in vitro motility assay showed that 30 μM EMD57033 and 100 μM Bepridil increase Ca(2+)-sensitivity of phosphorylated cardiac thin filaments by 3.1 and 2.8-fold respectively. Additionally they uncoupled Ca(2+)-sensitivity from TnI phosphorylation, mimicking the effect of HCM mutations. EGCG decreased Ca(2+)-sensitivity of phosphorylated and unphosphorylated wild-type thin filaments equally (by 2.15±0.45 and 2.80±0.48-fold respectively), retaining the coupling. Moreover, EGCG also reduced Ca(2+)-sensitivity of phosphorylated but not unphosphorylated thin filaments containing DCM and HCM-causing mutations, thus the dependence of Ca(2+)-sensitivity upon TnI phosphorylation of uncoupled mutant thin filaments was restored in every case. In single mouse heart myofibrils, EGCG reduced Ca(2+)-sensitivity of force and k(ACT) and also preserved coupling. Myofibrils from the ACTC E361G (DCM) mouse were uncoupled; EGCG reduced Ca(2+)-sensitivity more for phosphorylated than unphosphorylated myofibrils, thus restoring coupling. CONCLUSION: We conclude that it is possible to both mimic and reverse the pathological defects in troponin caused by cardiomyopathy mutations pharmacologically. Re-coupling by EGCG may be of potential therapeutic significance for treating cardiomyopathies

Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle

Background-Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca2+ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca2+-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation.Methods and Results-Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca2+-activated force was similar in cTnC G159D and donor myocytes, but the Ca2+ sensitivity of cTnC G159D myocytes was higher (EC50 G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca2+ sensitivity (EC(50)G159D/donor=0.55 +/- 0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca2+ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca2+ sensitivity was increased (EC50 P/unP=4.7 +/- 1.9) but not with cTnC G159D troponin (EC50 P/unP=1.2 +/- 0.1).Conclusions-We propose that uncoupling of the relationship between phosphorylation and Ca2+ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation. (Circ Heart Fail. 2009;2:456-464.

Contractile Dysfunction Irrespective of the Mutant Protein in Human Hypertrophic Cardiomyopathy With Normal Systolic Function

Background-Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. Methods and Results-Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3(mut); n = 17), mutation negative HCM patients without an identified sarcomere mutation (HCM(mn); n = 11), and nonfailing donors (n = 12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3(mut) by 33 +/- 5%, and similar in HCM(mn) compared with donor. cMyBP-C phosphory Conclusions-Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression. (Circ Heart Fail. 2012; 5: 36-46.

Rapidity and Centrality Dependence of Proton and Anti-proton Production from Au+Au Collisions at sqrt(sNN) = 130GeV

We report on the rapidity and centrality dependence of proton and anti-proton transverse mass distributions from Au+Au collisions at sqrt(sNN) = 130GeV as measured by the STAR experiment at RHIC. Our results are from the rapidity and transverse momentum range of |y|<0.5 and 0.35 <p_t<1.00GeV/c. For both protons and anti-protons, transverse mass distributions become more convex from peripheral to central collisions demonstrating characteristics of collective expansion. The measured rapidity distributions and the mean transverse momenta versus rapidity are flat within |y|<0.5. Comparisons of our data with results from model calculations indicate that in order to obtain a consistent picture of the proton(anti-proton) yields and transverse mass distributions the possibility of pre-hadronic collective expansion may have to be taken into account.Comment: 4 pages, 3 figures, 1 table, submitted to PR

The Fairy Tale Model:Secure Facility Therapist Perceptions

The current exploratory qualitative study sought to investigate novice therapist experience of implementing a phased trauma recovery approach, the Fairy Tale Model (FTM), in secure accommodation in Scotland. Participants were ten therapists trained and supervised in FTM over a 6 month period. Therapists delivered FTM to 37 youth. Individual interviews with therapists were based on the objectives of FTM, and explored the benefits, challenges and facilitating factors for both youth and therapists. Perceived benefits for therapists included increases in trauma-informed knowledge, skills, and confidence. Youth were perceived by therapists, to be less emotionally dysregulated and more motivated, hopeful, and communicative. Challenges for therapists involved the complexity of youth difficulties, competing work demands, difficulties unlearning established approaches, and short duration placements. Prioritizing therapy, intensive sessions, and frequent communication with care staff were seen as facilitating factors. Recommendations are made for FTM delivery and more robust mixed methods evaluative research including therapist, youth and other stakeholder perspectives

The National Lung Matrix Trial: translating the biology of stratification in advanced non-small-cell lung cancer

© The Author 2015.Background: The management of NSCLC has been transformed by stratified medicine. The National Lung Matrix Trial (NLMT) is a UK-wide study exploring the activity of rationally selected biomarker/targeted therapy combinations. Patients and methods: The Cancer Research UK (CRUK) Stratified Medicine Programme 2 is undertaking the large volume national molecular pre-screening which integrates with the NLMT. At study initiation, there are eight drugs being used to target 18 molecular cohorts. The aim is to determine whether there is sufficient signal of activity in any drug-biomarker combination to warrant further investigation. A Bayesian adaptive design that gives a more realistic approach to decision making and flexibility to make conclusions without fixing the sample size was chosen. The screening platform is an adaptable 28-gene Nextera next-generation sequencing platform designed by Illumina, covering the range of molecular abnormalities being targeted. The adaptive design allows new biomarker-drug combination cohorts to be incorporated by substantial amendment. The pre-clinical justification for each biomarker-drug combination has been rigorously assessed creating molecular exclusion rules and a trumping strategy in patients harbouring concomitant actionable genetic abnormalities. Discrete routes of pathway activation or inactivation determined by cancer genome aberrations are treated as separate cohorts. Key translational analyses include the deep genomic analysis of pre- and post-treatment biopsies, the establishment of patient-derived xenograft models and longitudinal ctDNA collection, in order to define predictive biomarkers, mechanisms of resistance and early markers of response and relapse. Conclusion: The SMP2 platform will provide large scale genetic screening to inform entry into the NLMT, a trial explicitly aimed at discovering novel actionable cohorts in NSCLC

Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics

Despite advancements in symptom management for heart failure (HF), this devastating clinical syndrome remains the leading cause of death in the developed world. Studies using animal models have greatly advanced our understanding of the molecular mechanisms underlying HF; however, differences in cardiac physiology and the manifestation of HF between animals, particularly rodents, and humans necessitates the direct interrogation of human heart tissue samples. Nevertheless, an ever-present concern when examining human heart tissue samples is the potential for artefactual changes related to temperature changes during tissue shipment or sample processing. Herein, we examined the effects of temperature on the post-translational modifications (PTMs) of sarcomeric proteins, the proteins responsible for muscle contraction, under conditions mimicking those that might occur during tissue shipment or sample processing. Using a powerful top-down proteomics method, we found that sarcomeric protein PTMs were differentially affected by temperature. Specifically, cardiac troponin I and enigma homolog isoform 2 showed robust increases in phosphorylation when tissue was incubated at either 4 °C or 22 °C. The observed increase is likely due to increased cyclic AMP levels and activation of protein kinase A in the tissue. On the contrary, cardiac troponin T and myosin regulatory light chain phosphorylation decreased when tissue was incubated at 4 °C or 22 °C. Furthermore, significant protein degradation was also observed after incubation at 4 °C or 22 °C. Overall, these results indicate that temperature exerts various effects on sarcomeric protein PTMs and careful tissue handling is critical for studies involving human heart samples. Moreover, these findings highlight the power of top-down proteomics for examining the integrity of cardiac tissue samples

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: A coupled electromechanical modeling study

Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa;L); and the enhancement in SERCA pump activity via phosphorylation of PLB.Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated -adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa;L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa;L as the key mechanisms underlying the aforementioned positive FFR

Azimuthal anisotropy and correlations in the hard scattering regime at RHIC

Azimuthal anisotropy (v(2)) and two-particle angular correlations of high p(T) charged hadrons have been measured in Au+Au collisions at roots(NN) = 130 GeV for transverse momenta up to 6 GeV/c, where hard processes are expected to contribute significantly. The two-particle angular correlations exhibit elliptic flow and a structure suggestive of fragmentation of high p(T) partons. The monotonic rise of v(2)(p(T)) for p(T) 3 GeV/c, a saturation of v(2) is observed which persists up to p(T) = 6 GeV/c